Nanosheres comprising a biocompatible polysaccharide

ABSTRACT

Microspheres, having a size lower than 1μ and comprising a biocompatible polysaccharidic polymer, are prepared with a process comprising the precipitation of polymer induced by means of a supercritical antisolvent (SAS). These microspheres are used as vehicling agents or carriers in the preparation of pharmaceutical compositions administrable by oral, nasal, pulmonary, vaginal or rectal route. These microspheres can also be advantageously used as vehicling agent or carriers in the preparation of pharmaceutical compositions for the treatment of human diseases associated with genic defects, for the preparation of diagnostics and in the agro-alimentary industry.

This is a division of application Ser. No. 08/938,288 filed Sep. 26,1997.

FIELD OF THE INVENTION

The present invention relates to:

microspheres having a diameter ≧0.1 and <1μ, comprising a biocompatiblepolysaccharide polymer and optionally at least one active ingredient,

pharmaceutical compositions containing said microspheres administrableby oral, nasal, pulmonary, vaginal or rectal route,

the use of microspheres having a diameter ranging from 0.1 to 1μ ascarriers for the preparation of pharmaceutical compositions for humangenic therapy, for the preparation of diagnostics and in theagroalimentary industry,

a process for the preparation of microspheres having a dimension ofbetween 0.1 and 1μ comprising the precipitation of said polymer inducedby means of a supercritical antisolvent (SAS).

TECHNOLOGICAL BACKGROUND

Major advances have recently been made in pharmaceutical technology toresearch new methods for the preservation of the intrinsic activity ofpolypeptides and to render them absorbent. Formulations able to ensure areproducible absorption of these active molecules have the advantage oflacking side effects, unlike synthetic polymers. Of all the most widelyused natural polymers, the category of acidic polysaccharides is ofparticular interest. One of these, hyaluronic acid, a polysaccharidewidely distributed throughout animal organisms, is constituted by unitsof D-glucuronic acid and N-acetyl D-glucosamine in alternate order. Itsmolecular weight can vary according to the methods used for itsextraction and/or purification (EP 0138572 reg. on Jul. 25, 1990: EPA0535200 published on Apr. 7, 1993; PCT Application No. WO 95/04132published on Feb. 9, 1995; PCT Patent Application No. WO 95/24497published on Sep. 14, 1995).

Besides the polymer's chemical-physical properties, the release methodsand systems for biologically active molecules are also particularlyimportant, such as microspheres which seem to be among the mostversatile release systems. EPA 0517565 discloses a process for thepreparation of microspheres, whose dimensions range between 1-100 μm,wherein the polysaccharide ester dissolved in an aprotic solvent such asDMSO, is added to a mixture of a high-viscosity mineral oil containing anon ionic surface active agent and ethyl acetate, which is a solvent forDMSO and the mineral oil, but not for the polysaccharide ester, whichtherefore precipitates in the form of microspheres having therefore theabove mentioned dimensions.

Today, various techniques are known which involve the use ofsupercritical fluids for the production of finely subdivided particleswith a narrow granulometric distribution curve. The supercriticalantisolvent process is generally performed at moderate temperatures andenables the solvent to be completely removed from the precipitationenvironment. The applications concern substances that are heat-sensitiveor difficult to handle, such as explosives (Gallagher, P. M. et al.,1989, Supercritical Fluid Science and Technology—Am. Chem. Soc.334-354). Other applications concern the production of polymers in theform of fibers (Dixon, D. J. et al, 1993, J. Appl. Polym. Sci. 50,1929-1942) and in the form of microparticles, including microspheres(Dixon, D. J., et al., 1993, AIChE J., 39, 1, pp 127-139). In thepharmaceutical field, the main interest is in the treatment of proteins(Tom, J. W., et al, 1994, Supercritical Fluid Engineering Science, pp238-257, ACS Symp. Chap. 19, Ed. H. Kiran and J. F. Brennecke; Yeo, S.D., et al, 1993, Biotech. and Bioeng., 41, pp 341-346) and biodegradablepolymers, such as poly(L-lactic acid) (Randolph, T. W., et al, 1993,Biotechnol. Prog., 9, 429-435; Yeo, S. D., et al, 1993, Macromolecules,26, 6207-6210). Various methods have been devised for precipitation witha supercritical antisolvent. The semi-discontinuous method (Gallagher etal., 1989), involves injection of the antisolvent in the liquid solutionwhich has already been prepared in the desired working conditions. Theoperation must be performed in a stepwise fashion to ensure that theliquid is removed, the final quantities of product are very limited andthe spheres measure far more than 1μ in size.

Precipitation with a compressed antisolvent (PCA) involves injection ofthe solution in the high-density supercritical fluid (SCF) (Dixon etal., 1991; Dixon and Johnston, 1993). The injection times are muchreduced to guarantee complete dissolution of the liquid, so the quantityof precipitate is very low, giving microfibers with an orderedstructure.

The continuous process (Yeo et al., 1993a) enables the solution and theantisolvent to be injected simultaneously in the precipitationenvironment: the liquid expands and evaporates in the continuous phase,constituted by the SCF. The solution is injected through a micrometricnozzle with a diameter ranging between 10 and 30μ. Solutions must bediluted to avoid blocking the nozzle and to present reticulatestructures being formed. Consequently, the quantity of solid soluteinjected is very low. Moreover, a high ratio between the volume ofantisolvent and solution must be used to continuously remove the liquidsolvent from the precipitation vessel.

When the solution is placed in the precipitator and the container isloaded by means of SCF up to the desired pressure, the process assumes acompletely discontinuous character (Yeo et al., 1993 a,b). By thistechnique, microspheres with a diameter of over 1μ have been obtained.All the methods described here are accompanied by a final washing stepto prevent the precipitate being resolubilized by the solvent. However,none of the cited techniques has been specifically applied to theproduction of high-molecular-weight biocompatible polysaccharidepolymers and in particular the HYAFFs, namely the ester of hyaluronicacid, which are obtained by the procedure described in U.S. Pat. No.4,851,521.

SUMMARY OF THE INVENTION

The Applicant has unexpectedly found that with the discontinuous SAStechnique it is possible to obtain in quantitative yields microsphereswith a diameter of less than 1μ comprising an ester of a biocompatibleacidic polysaccharide polymer, selected from the group consisting of:hyaluronic acid esters, crosslinked esters of hyaluronic acid, esters ofchitin, esters of pectin, esters of gellan, esters of alginic acid.

Object of the present invention are therefore microspheres having adimension ≧0.1μ and <1μ comprising a biocompatible polysaccharidepolymer.

A further object of the present invention are pharmaceuticalcompositions administrable by oral, nasal, pulmonary, vaginal or rectalroute, containing said microspheres as vehicling agents or carriers incombination with at least one active ingredient and optionally withfurther conventional excipients.

A further object of the present invention relates to said microspheresfurther comprising at least one of the following active principles: apharmaceutically active polypeptide, a Granulocyte Macrofage ColonyStimulating Factor (GMCSF), a trophic factor, an immunoglobulin, anatural or a synthetic derivative of a ganglioside, an antiviral, anantiasthmatic an antiinflammatory agent, an antibiotic and anantimycotic agent.

A further object of the present invention relates to pharmaceuticalcompositions administrable by oral, nasal, pulmonary, vaginal or rectalroute containing the microspheres inglobating the above mentioned activeprinciples, optionally in combination with other conventionalexcipients.

A further object of the present invention relates to the use of saidmicrospheres as carriers in the preparation of diagnostics and inagroalimentary industry. Moreover the microspheres having a diameterranging from 0.1 to 1μ containing a biocompatible acidic polysaccharideester selected from the group consisting of: hyaluronic acid esters,esters of chitin, esters of pectin, esters of gellan, esters of alginicacid can be advantageously used as vehicling agent or carriers of agene, for the preparation of pharmaceutical compositions for thetreatment of diseases associated with genic defects.

A further object of the present invention resides in the discontinuousprocess for the preparation of microspheres having a dimension comprisedbetween 0.1 and 1μ and comprising the precipitation of said polymerinduced by means of a supercritical antisolvent (SAS). The processobject of the present invention comprises the following steps:

a) dissolving the polysaccharide biocompatible polymer in an aproticsolvent at concentrations ranging from 0.1 to 5% by weight,

b) charging the solution of step (a) in a pressure proof containerhaving at the top and at the base steel filters with an average cut-offlower than 0.1μ;

c) loading from underneath the antisolvent until reaching the pressureat which said fluid becomes supercritical at a temperature ranging from25 to 60° C.,

d) removing the aprotic solvent, by flowing said supercritical fluid,

e) depressurizing the pressure proof container and collecting theprecipitated product.

Contrarily to what one could foresee from the above mentioned prior art(teaching that, with the SAS discontinuous technique, process times arelonger than with the continuous one, nucleation occurs in the bulkliquid phase where the supercritical antisolvent is dissolved andtherefore the formation of large particles with broad granulometricdistribution is expected), surprisingly the expanding conditions adoptedwith the process according to the present invention enable the onset ofthe nucleation process in a well-expanded media so that the formation ofa high number of nucleation centres is achieved. This factor, combinedwith the amorphous nature of the solid solute, leads to the formation ofmicrospheres whose dimension is comprised in the above mentioned rangeand moreover with a narrow granulometric distribution curve.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 represents a SEM photograph (Scanning Electron Microscope) ofHYAFF-11 microspheres obtained by following the operating conditionsreported in Example 1, starting from a HYAFF concentration in DMSO equalto 1% w/w (bar=1 micron);

FIG. 2 is a photograph of the sample relative to FIG. 1 with a highermagnification (bar=1 micron);

FIG. 3 represents a SEM photograph of HYAFF-11 p75 microspheres obtainedaccording to the working conditions of Example 2, starting from a HYAFFconcentration in DMSO equal to 1% w/w (bar=1 micron);

FIG. 4 represents a SEM photograph of HYAFF-7 microspheres prepared byfollowing the operating conditions described in Example 3, starting froma HYAFF concentration in DMSO equal to 1% w/w (bar=1 micron);

FIG. 5 represents a SEM photograph of ACP p10 microspheres obtained byfollowing the operating conditions described in Example 4, starting froman ACP concentration in DMSO equal to 1% w/w (bar=1 micron);

FIG. 6 represents a SEM photograph of ALAFF microspheres, prepared byfollowing the operating conditions described in Example 5 starting froman ALAFF concentration in DMSO equal to 1% w/w (bar=1 micron).

DETAILED DESCRIPTION OF THE INVENTION

The biocompatible polysaccharide polymer which is comprised in themicrospheres according to the present invention is preferably an esterof a polysaccharide acid such a hyaluronic acid ester, selected fromthose described in U.S. Pat. No. 4,851,521, which we incorporate byreference, a crosslinked ester of hyaluronic acid selected from thosedisclosed in EP 0341745 E1 which we incorporate by reference, an esterof chitin selected from those described in PCT WO93/06136, which weincorporate by reference, an ester of pectin selected from thosementioned in PCT WO93/14129, which we incorporate by reference, an esterof gellan selected from those disclosed in U.S. Pat. No. 5,332,809,which we incorporate by reference, an ester of alginic acid selectedfrom those reported in U.S. Pat. Nos. 5,264,422 and 5,336,668, which weincorporate by reference. Particularly preferred esters are the total orpartial benzyl ester of hyaluronic acid. Among the partial ester aparticularly preferred ester is the benzyl ester with 75% of the carboxyfunction of hyaluronic acid esterified with benzyl alcohol.

The pharmaceutical compositions according to the present inventioncontaining said microspheres as vehicling agents or carriers, incombination with at least one active agent can optionally be formulatedin a controlled release form, in order to have the desired rate ofabsorption, with suitable excipients normally used for preparing thistype of formulations.

Preferred pharmaceutically active polypeptides which can be comprised inthe microspheres according to the present invention are calcitonin,insulin, preferred trophic factors, which can be incorporated in themicrospheres according to the present invention are the Nerve GrowthFactor (h-NGF), the Ciliary Neuronotrophic Growth Factor (h-CNTF).

The pharmaceutical compositions containing the above microspheresincorporating the above listed active principles, can optionally beformulated in controlled release form, in order to have the desired rateof absorption, with suitable excipients normally used for preparing thistype of formulations.

As pointed out above the microspheres having a diameter ≧0.1μ and <1μcan be advantageously used as vehicling agents in the preparation ofdiagnostics. In particular, according to the type of technique to beused for diagnostic analysis, such as NMR, ultrasound, X rays, themicrospheres can be loaded with paramagnetic agents such as magnetite,or they may be concave in structure, or, alternatively, they may beloaded with nonionic contrast agents, or, lastly, with radioactiveisotopes such as TC^(99m).

As a matter of face vehicling of the contrast agents by means ofmicrospheres limits interaction with the blood, thus reducing the onsetof the side effects typically caused by contrast agents.

As previously pointed out, another important sector in which themicrospheres having a diameter comprised between 0.1 and 1μ according tothe present invention can be advantageously used is the preparation ofpharmaceutical compositions for the treatment of diseases associatedwith genic defects.

Much effort is currently being put into scientific research in thisfield to find remedies for genetic-type malformations or metabolicdiseases of a genetic origin. Most of the work being done is aimed atidentifying and preparing vehicling systems for healthy genetic materialto be administered to patients suffering from such malformations anddiseases. One of the possibilities is represented by the encapsulationof healthy genes in microspheres which are able to penetrate more deeplyinto the tissues and sustain contact with the cell surfaces to betreated for longer periods of time. It follows that the adherence of themicrospheres to the cell surfaces enables the release of geneticmaterial transported to the close vicinity of the target cells. Inparticular, the microspheres having a diameter ranging from 0.1 to 1μcontaining the biocompatible polysaccharide polymer according to thepresent invention represent an ideal transport system for biologicalmaterial, and in this particular case for healthy genes, thanks to theirvery small dimensions and specific mucoadhesiveness. Among the possibleapplications for said microspheres in the treatment of human diseasesassociated with genic defects a preferred one is in their use asvehicling agents of single genes which encode specific enzymes, for thetreatment of diseases caused by a deficit of the same enzymes. There arein fact numerous diseases which derive from an enzyme deficit orhyperactivity, which is caused by defects occurred in the specific geneencoding this enzyme.

For example diseases of this type are:

phenylketonuria, due to a deficit of phenylalanine hydroxylase,

alkaptonuria, due to a deficit of homogentisic acid oxidase,

albinism due to a deficit of tyrosinase and many other diseasesinvolving amino acid metabolism;

diseases involving glycogen accumulation, some of which are fatal atbirth, due to deficit of enzymes such as glucose-6-phosphatase, brancheror de-brancher enzymes, and α-lysosomal glucosidase enzymes;

carbohydrate metabolism disorders

Wilson's disease, involving a defect in ceruloplasma, the protein whichtransports copper

porphyria caused by a deficit in porphobilinogen deaminase,uroporphyrinogen oxydase, protoporphyrinogen oxydase coproporphyrinogenoxydase,

gout due to hypoxanthine-guanine-phosphoribosyl transferase deficiency,or hyperactivity of 5-phosphoribosyl-1-pyrophosphate transferase,

diseases involving lysosomal accumulation such as gangliosidosis, due toβ-galactosidase deficiency, leukodystrophy, Niemann-Pick's disease dueto sphingomyelinase deficiency, Gaucher's disease due toglucosyl-ceramidase deficiency, Fabry's disease, due to α-galactosidasedeficiency, mucopolysaccharidosis etc.,

connective tissue disorders (brittle bone syndrome, Ehlers-Danlossyndrome, Marfan syndrome).

Besides their use in enzymatic deficits, the microspheres can be used tovehicle single genes in any pathologies wherein such genes are altered,such as malformative diseases of genetic origin (Down's syndrome,arachnodactyly etc.), hereditary diseases such as:

hemoglobinopathies (sickle-cell anaemia, thalassaemia etc),

cystic fibrosis,

primitive hyperlipoproteinemia and other lipid metabolism disorders,wherein single or multifactorial gene disorders with hereditarytransmission and complex modalities of different genes, interact withenvironmental factors, thus determining hyperlipoproteinemia having adifferent degree of seriousness in different members of the same family,

cancer wherein it has been ascertained that genetic alterations exist atthe level of the differentiation and of the failed control of cellulargrowth.

Finally as pointed out above, the microspheres having a diameter ≧0.1μand <1μ can be advantageously used in the agro-alimentary sector, forexample as a vehicle for plant treatments or for the preservation ofadditives.

The preferred supercritical fluid used as antisolvent in the processaccording to the present invention is selected from carbon dioxide (CO₂)and hydrofluorocarbons, such as trifluoromethane.

In this specific case when CO₂ in step (c) it is charged with a loadingrate or pressure gradient ranging from 3 to 20 bar/min, preferably 10bar/min, until a pressure is reached in the pressure proof containerranging from 80 to 120 bar/min, more preferably 100 bar/min.

Precipitation of the polymer in this step is induced by thesupercritical antisolvent which, by solubilizing and expanding thesolution, causes a decrease in the solvent power of the liquid andsimultaneous evaporation. The dissolved product, not soluble in the SCF,separates as a solid.

The particles in step (d) are washed with the antisolvent to remove theliquid completely before the precipitator is depressurized.

The depressurization in step (e) of the process according to the presentinvention is preferably carried out by using a pressure gradient of 5bar/min.

The preferred solvent used in step (a) to dissolve the biocompatiblepolysaccharide polymer is selected from dimethylsulfoxide andN-methylpyrrolidone.

The microspheres according to the present invention further comprisingat least one of the above mentioned active principles can be prepared intwo alternative ways.

The first one encompasses the addition of the active principle in step(a) of the process according to the present invention, after thedissolution of the biocompatible polysaccharide polymer in the aproticsolvent.

The coprecipitation of the active principle in step (c) with thebiocompatible polysaccharide polymer does not alter the form ormorphology of the precipitate.

According to the latter way, the microspheres coming from step (e), aresuspended in a buffered solution preferably a phosphate buffer solutioncontaining the desired active principle at a suitable concentration inorder to obtain the desired active ingredient titer/mg of microsphere,and the suspension is subjected to liophylization at the liquid nitrogentemperature.

We report hereafter, for purely illustrative purposes, some examples ofhow to obtain microspheres made with polymer alone with polymercontaining pharmacologically active substances. Any variations whichwould be obvious to an expert in the field are to be considered ascoming within the scope of the present invention.

EXAMPLE 1

Preparation of microspheres within the starting polymer is HYAFF-11(benzyl ester of hyaluronic acid)

A hyaluronic acid ester, wherein all the carboxy groups of hyaluronicacid are esterified with benzyl alcohol, is dissolved in an aproticsolvent, such as dimethylsulfoxide (DMSO), at a concentration varyingbetween 0.1 and 5% in weight, generally 1% w/w. Once the polymer hassolubilized, the solution is poured into a pressure-proof container(precipitator), thermostatically controlled with a heated ethyleneglycol jacket. Porous steel filters with an average cut-off of less than0.1μ are screwed onto the base and top of the precipitator.

The liquid is unable to seep through by gravity alone. Once thecontainer is closed, it is loaded from underneath with hyperpure carbondioxide (CO₂) until the working pressure is reached (80-120 bar,preferably 100 bar). The CO₂ is dispersed in the solution through thefilter. This antisolvent, which is first gaseous and then supercritical,can be mixed perfectly with the liquid solvent (DMSO) but it is anonsolvent for the polymer.

The loading rate, or the pressure gradient over time, is set in a rangeof 3-20 bar/min, preferably 10 bar/min. The temperature in theprecipitator is kept constant in a range of between 25° C. and 60° C.,preferably 40° C.

When the working pressure has been reached, the flow of CO₂ is switchedoff for 10 minutes to obtain the desired pressure and temperatureconditions inside the precipitator. The washing operation is begun bysupplying antisolvent to the precipitator and regulating the outlet flowfrom the top of the precipitator by means of a millimetric valve.

The outlet fluid, constituted by antisolvent and DMSO, is directedtowards the DMSO collector, which is kept at room pressure; the DMSOseparates after expansion and consequent cooling, while the gaseous CO₂comes out of the top of the container and is released into theatmosphere. The solid particles, on the other hand, are trapped by theporous filters at the top and base of the precipitator.

The operation is continued to allow the DMSO to be completely removedfrom the precipitator. The time it takes for the organic solvent to beremoved by the supercritical antisolvent depends on the temperature inthe precipitation chamber, when fixed amount of liquid solution anantisolvent flow rate are set up.

At the end of washing, the supply of CO₂ is cut off and the container isdepressurized at a rate of 5 bar/min. The container is opened, themicrospheres are collected and placed in suitable containers where theyare stored at 4° C. The yield of microspheres is almost total. There isno appreciable incorporation of solvent in the precipitate. The DMSO iscollected in the expansion container.

The mean particle size in these working conditions is 0.6μ (FIG. 12).

EXAMPLE 2

Preparation of microspheres wherein the starting polymer is HYAFF-11 p75(partial benzyl ester of hyaluronic acid)

A hyaluronic acid ester, wherein 75% of the carboxy groups of hyaluronicacid are esterified with benzyl alcohol, while the remaining part issalified with sodium, is dissolved in an aprotic solvent such asdimethylsulfoxide (DMSO), at a concentration varying between 0.1 and 5%in weight, generally 1% w/w. Once the polymer has reachedsolubilization, the solution is poured into a pressure-proof container(precipitator), thermostatically controlled by a heated ethylene glycoljacket. Porous steel filters with a cut-off of 0.1μ are screwed onto thetop and base of the precipitator. The liquid is unable to seep throughby gravity alone.

Once the vessel is closed, it is loaded from underneath with hyperpurecarbon dioxide (CO₂) until the working pressure is reached (80-120 bar,preferably 100 bar). The CO₂ is distributed in the solution through theporous filter. This antisolvent, which is first gaseous and thensupercritical, can be mixed perfectly with the liquid solvent (DMSO) butit is a nonsolvent for the polymer.

The loading rate, or the pressure gradient over time, is set in a rangeof 3-20 bar/min, preferably 10 bar/min. The temperature in theprecipitator is kept constant in a range of between 25° C. and 60° C.,preferably 40° C.

When the working pressure has been reached, the flow of CO₂ is switchedoff for 10 minutes to obtain the desired pressure and temperatureconditions inside the precipitator. The washing operation is begun bysupplying antisolvent to the precipitator and regulating the outlet flowfrom the top of the precipitator by means of a millimetric valve.

The outlet fluid, constituted by antisolvent and DMSO, is directedtowards the DMSO collector, which is kept at room pressure; the DMSOseparates after expansion and consequent cooling, while the gaseous CO₂comes out of the top of the vessel and is dispersed in the atmosphere.The solid particles, on the other hand, are trapped by the porousfilters at the top and bottom of the precipitator.

The operation is continued to allow the DMSO to be completely removedfrom the precipitator. The time it takes for the organic solvent to beremoved by the supercritical antisolvent depends on the temperature inthe precipitation chamber, when fixed amount of liquid solution andantisolvent flow rate are set up.

At the end of washing, the supply of CO₂ is cut off and the vessel isdepressurized at a rate of 5 bar/min. The vessel is opened, themicrospheres are collected and placed in suitable containers where theyare stored at 4° C. The yield of microspheres is almost total. There isno appreciable incorporation of solvent in the precipitate. The DMSO iscollected in the expansion container.

The mean particle size in these working conditions is 0.8μ (FIG. 3).

EXAMPLE 3

Preparation of microspheres wherein the starting polymer is HYAFF-7(ethyl ester of hyaluronic acid)

A hyaluronic acid ester, wherein all the carboxy groups of hyaluronicacid are esterified with ethyl alcohol, is dissolved in an aproticsolvent such as dimethylsulfoxide (DMSO), at a concentration varyingbetween 0.1 and 5% in weight, generally 1% w/w. Once the polymer hasreached solubilization, the solution is poured into a pressure-proofvessel (precipitator), thermostatically controlled by a heated ethyleneglycol jacket. Porous steel filters with a cut-off of 0.1μ are screwedonto the top and bottom of the precipitator. The liquid is unable toseep through by gravity alone.

Once the vessel is closed, it is loaded from underneath with hyperpurecarbon dioxide (CO₂) until the working pressure is reached (80-120 bar,preferably 100 bar). The CO₂ is distributed in the solution through theporous base. This antisolvent, which is first gaseous and thensupercritical, can be mixed perfectly with the liquid solvent (DMSO) butit is a nonsolvent for the polymer.

The loading rate, or the pressure gradient over time, is set in a rangeof 3-20 bar/min, preferably 10 bar/min. The temperature in theprecipitator is kept constant in a range of between 25° C. and 60° C.,preferably 40° C.

When the working pressure has been reached, the flow of CO₂ is switchedoff for 10 minutes to obtain the desired pressure and temperatureconditions inside the precipitator. The washing operation is begun bysupplying antisolvent to the precipitator and regulating the outlet flowfrom the top of the precipitator by means of a millimetric valve.

The outlet fluid, constituted by antisolvent and DMSO, is directedtowards the DMSO collector, which is kept at room pressure; the DMSOseparates after expansion and consequent cooling, while the gaseous CO₂comes out of the top of the vessel and is released into the atmosphere.The solid particles, on the other hand, are trapped by the porousfilters at the top and base of the precipitator.

The operation is continued to allow the DMSO to be completely removedfrom the precipitator. The time it takes for the organic solvent to beremoved by the supercritical antisolvent depends on the temperature inthe precipitation chamber, when fixed amount of liquid solution andantisolvent flow rate are set up.

At the end of washing, the supply of CO₂ is cut off and the vessel isdepressurized at a rate of 5 bar/min. The vessel is opened, themicrospheres are collected and placed in suitable containers where theyare stored at 4° C. The yield of microspheres is almost total. There isno appreciable incorporation of solvent in the precipitate. The DMSO iscollected in the expansion container.

The mean particle size in these working conditions is 1.0μ (FIG. 4).

EXAMPLE 4

Preparation of microspheres wherein the starting polymer is acrosslinked polysaccharide of hyaluronic acid (ACP)

A hyaluronic acid derivative, wherein 10% of the carboxy groups ofhyaluronic acid are bound with inter- or intramolecular hydroxy groupsand the remaining part is salified with sodium, is dissolved in anaprotic solvent such as dimethylsulfoxide (DMSO), at a concentrationvarying between 0.1 and 5% in weight, generally 1% w/w. The proceduredescribed in Example 1 is then performed. The mean particle size is 0.6μ(FIG. 5).

EXAMPLE 5

Preparation of microspheres wherein the starting polymer is an ester ofalginic acid (ALAFF)

A derivative of alginic acid, wherein all the carboxy groups of alginicacid are esterified with benzyl alcohol, is dissolved in an aproticsolvent, such as dimethylsulfoxide (DMSO), at a concentration varyingbetween 0.1 and 5% in weight, generally 1% w/w. The procedure describedin Example 1 is then performed. The mean particle size is 0.8μ (FIG. 6).

EXAMPLE 6

Preparation of microspheres wherein the starting polymer is an ester ofpectinic acid

A derivative of pectinic acid, wherein all the carboxy groups areesterified with benzyl alcohol, is dissolved in an aprotic solvent, suchas dimethylsulfoxide (DMSO), at a concentration varying between 0.1 and5% in weight, generally 1% w/w. The procedure described in Example 1 isthen performed.

The mean particle size is 0.7μ.

EXAMPLE 7

Preparation of microspheres wherein the starting polymer is HYAFF-11(benzyl ester of hyaluronic acid) and which are loaded with calcitonin

A hyaluronic acid ester, wherein all the carboxy groups of hyaluronicacid are esterified with benzyl alcohol, is dissolved in an aproticsolvent such as dimethylsulfoxide (DMSO), at a concentration varyingbetween 0.1% and 5% in weight, generally 1% w/w. Once the polymer hasreached solubilization, the calcitonin is added to the polymericsolution at the set concentration, eg 1.5 I.U. per mg of polymer.

The solution thus obtained is poured into a pressure-proof vessel(precipitator), thermostatically controlled by a heated ethylene glycoljacket. Porous steel filters with a cut-off of 0.1μ are screwed onto thetop and base of the precipitator. The liquid is unable to seep throughby gravity alone.

Once the vessel is closed, it is loaded from underneath with hyperpurecarbon dioxide (CO₂) until the working pressure is reached (80-120 bar,preferably 100 bar). The CO₂ is distributed in the solution through theporous base. This antisolvent, which is first gaseous and thensupercritical, can be mixed perfectly with the liquid solvent (DMSO) butit is a nonsolvent for the polymer and the polypeptide calcitonin.

The loading rate, or the pressure gradient over time, is set in a rangeof 3-20 bar/min, preferably 10 bar/min. The temperature in theprecipitator is kept constant in a range of between 25° C. and 60° C.,preferably 40° C.

When the working pressure has been reached, the flow of CO₂ is switchedoff for 10 minutes to obtain the desired pressure and temperatureconditions inside the precipitator. The washing operation is begun bysupplying antisolvent to the precipitator and regulating the outlet flowfrom the top of the precipitator by means of a millimetric valve.

The outlet fluid, constituted by antisolvent and DMSO, is directedtowards the DMSO collector, which is kept at room pressure; the DMSOseparates after expansion and consequent cooling, while the gaseous CO₂comes out of the top of the vessel and is released into the atmosphere.The solid particles, on the other hand, are trapped by the porousfilters at the top and base of the precipitator.

The operation is continued to allow the DMSO to be completely removedfrom the precipitator. The time it takes for the organic solvent to beremoved by the supercritical antisolvent depends on the temperature inthe precipitation chamber, when fixed amount of liquid solution andantisolvent flow rate are set up.

At the end of washing, the supply of CO₂ is cut off and the vessel isdepressurized at a rate of 5 bar/min. The vessel is opened, themicrospheres are collected and placed in suitable containers where theyare stored at 4° C. The yield of microspheres is almost total. There isno appreciable incorporation of solvent in the precipitate. The DMSO iscollected in the expansion container.

The mean particle size in these working conditions is 0.5μ. The quantityof incorporated calcitonin is 1.3 I.U. per mg of microspheres.

EXAMPLE 8

Preparation of microspheres wherein the starting polymer is HYAFF-11 p75(benzyl ester of hyaluronic acid) and which are loaded with calcitonin

A hyaluronic acid ester, wherein 75% of the carboxy groups of hyaluronicacid are esterified with benzyl alcohol, while the remaining part issalified with sodium, is dissolved in an aprotic solvent such asdimethylsulfoxide (DMSO), at a concentration varying between 0.1 and 5%in weight, generally 1% w/w. Once the polymer has reachedsolubilization, calcitonin is added to the polymeric solution at a setconcentration, eg 1.0 I.U. per mg of polymer.

The solution thus obtained is poured into a pressure-proof vessel(precipitator), thermostatically controlled by a heated ethylene glycoljacket. Porous steel filters with a cut-off of 0.1μ are screwed onto thetop and base of the precipitator. The liquid is unable to seep throughby gravity alone.

Once the vessel is closed, it is loaded from underneath with hyperpurecarbon dioxide (CO₂) until the working pressure is reached (80-120 bar,preferably 100 bar). The CO₂ is distributed in the solution through theporous filter. This antisolvent, which is first gaseous and thensupercritical, can be mixed perfectly with the liquid solvent (DMSO) butit is a nonsolvent for the polymer and the polypeptide calcitonin.

The loading rate, or the pressure gradient over time, is set in a rangeof 3-20 bar/min, preferably 10 bar/min. The temperature in theprecipitator is kept constant in a range of between 25° C. and 60° C.,preferably 40° C.

When the working pressure has been reached, the flow of CO₂ is switchedoff for 10 minutes to obtain the desired pressure and temperatureconditions inside the precipitator. The washing operation is begun bysupplying antisolvents to the precipitator and regulating the outletflow from the top of the precipitator by means of a millimetric valve.

The outlet fluid, constituted by antisolvent and DMSO, is directedtowards the DMSO collector, which is kept at room pressure; the DMSOseparates after expansion and consequent cooling, while the gaseous CO₂comes out of the top of the vessel and is released into the atmosphere.The solid particles, on the other hand, are trapped by the porousfilters at the top and base of the precipitator.

The operation is continued to allow the DMSO to be completely removedfrom the precipitator. The time it takes for the organic solvent to beremoved by the supercritical antisolvent depends on the temperature inthe precipitation chamber, when fixed amount of liquid solution andantisolvent flow rate are set up.

At the end of washing, the supply of CO₂ is cut off and the vessel isdepressurized at a rate of 5 bar/min. The vessel is opened, themicrospheres are collected and placed in suitable containers where theyare stored at 4° C. The yield of microspheres is almost total. There isno appreciable incorporation of solvent in the precipitate. The DMSO iscollected in the expansion container.

The mean particle size in these working conditions is 0.8μ. The quantityof incorporated calcitonin is 0.9 I.U. per mg of microspheres.

EXAMPLE 9

Preparation of microspheres wherein the starting polymer is HYAFF-7(ethyl ester) of hyaluronic acid, and which are loaded with calcitonin

A hyaluronic acid ester, wherein all the carboxy groups of hyaluronicacid are esterified with ethyl alcohol, is dissolved in an aproticsolvent such as dimethylsulfoxide (DMSO), at a concentration varyingbetween 0.1 and 5% in weight, generally 1% w/w. Once the polymer hasreached solubilization, calcitonin is added to the polymeric solution ata set concentration, eg 15 I.U. per mg of polymer.

The solution thus obtained is poured into a pressure-proof vessel(precipitator), thermostatically controlled by a heated ethylene glycoljacket. Porous steel filters with a cut-off of 0.1μ are screwed onto thetop and base of the precipitator. The liquid is unable to seep throughby gravity alone.

Once the vessel is closed, it is loaded from underneath with hyperpurecarbon dioxide (CO₂) until the working pressure is reached (80-120 bar,preferably 100 bar). The CO₂ is distributed in the solution through theporous base. This antisolvent, which is first gaseous and thensupercritical, can be mixed perfectly with the liquid solvent (DMSO) butit is a nonsolvent for the polymer and the polypeptide calcitonin.

The loading rate, or the pressure gradient over time, is set in a rangeof 3-20 bar/min, preferably 10 bar/min. The temperature in theprecipitator is kept constant in a range of between 25° C. and 60° C.,preferably 40° C.

When the working pressure has been reached, the flow of CO₂ is switchedoff for 10 minutes to obtain the desired pressure and temperatureconditions inside the precipitator. The washing operation is begun bysupplying antisolvent to the precipitator and regulating the outlet flowfrom the top of the precipitator by means of a millimetric valve.

The outlet fluid, constituted by antisolvent and DMSO, is directedtowards the DMSO collector, which is kept at room pressure; the DMSOseparates after expansion and consequent cooling, while the gaseous CO₂comes out of the top of the vessel and is released into the atmosphere.The solid particles, on the other hand, are trapped by the porousfilters at the top and bottom of the precipitator.

The operation is continued to allow the DMSO to be completely removedfrom the precipitator. The time it takes for the organic solvent to beremoved by the supercritical antisolvent depends on the temperature inthe precipitation chamber, when fixed amount of liquid solution andantisolvent flow rate are set up.

At the end of washing, the supply of CO₂ is cut off and the vessel isdepressurized at a rate of 5 bar/min. The vessel is opened, themicrospheres are collected and placed in suitable containers where theyare stored at 4° C. The yield of microspheres is almost total. There isno appreciable incorporation of solvent in the precipitate. The DMSO iscollected in the expansion container.

The mean particle size in these working conditions is 1.0μ. The quantityof incorporated calcitonin is 13 I.U. per mg of microspheres.

EXAMPLE 10

Preparation of microspheres wherein the starting polymer is HYAFF-11(benzyl ester of hyaluronic acid), and which contain GMCSF (granulocytemacrophage colony stimulating factor).

A hyaluronic acid ester, wherein all the carboxy groups of hyaluronicacid are esterified with benzyl alcohol, is dissolved in an aproticsolvent such as dimethylsulfoxide (DMSO), at a concentration whichvaries between 0.1 and 5% in weight, generally 1% w/w. Once the polymerhas reached solubilization. GMCSF is added to the polymer solution at aset concentration, eg 1% of the polymer mass.

The solution thus obtained is poured into a pressure-proof vessel(precipitator), thermostatically controlled by a heated ethylene glycoljacket. Porous steel filters with a cut-off of 0.1μ are screwed onto thetop and base of the precipitator. The liquid is unable to seep throughby gravity alone.

Once the vessel is closed, it is loaded from underneath with hyperpurecarbon dioxide (CO₂) until the working pressure is reached (80-120 bar,preferably 100 bar). The CO₂ is distributed in the solution through theporous base. This antisolvent, which is first gaseous and thensupercritical, can be mixed perfectly with the liquid solvent (DMSO) butit is a nonsolvent for the polymer and the polypeptide GMCSF.

The loading rate, or the pressure gradient over time, is set in a rangeof 3-20 bar/min, preferably 10 bar/min. The temperature in theprecipitator is kept constant in a range of between 25° C. and 60° C.,preferably 40° C.

When the working pressure has been reached, the flow of CO₂ is switchedoff for 10 minutes to obtain the desired pressure and temperatureconditions inside the precipitator. The washing operation is begun bysupplying antisolvent to the precipitator and regulating the outlet flowfrom the top of the precipitator by means of a millimetric valve.

The outlet fluid, constituted by antisolvent and DMSO, is directedtowards the DMSO collector, which is kept at room pressure; the DMSOseparates after expansion and consequent cooling, while the gaseous CO₂comes out of the top of the vessel and is released into the atmosphere.The solid particles, on the other hand, are trapped by the porousfilters at the top and base of the precipitator.

The operation is continued to allow the DMSO to be completely removedfrom the precipitator. The time it takes for the organic solvent to beremoved by the supercritical antisolvent depends on the temperature inthe precipitation chamber, when fixed amount of liquid and antisolventflow rate are set up.

At the end of washing, the supply of CO₂ is cut off and the vessel isdepressurized at a rate of 5 bar/min. The vessel is opened, themicrospheres are collected and placed in suitable containers where theyare stored at 4° C. The yield of microspheres is almost total. There isno appreciable incorporation of solvent in the precipitate. The DMSO iscollected in the expansion container.

The mean particle size in these working conditions is 0.5μ. The quantityof incorporated GMCSF is 9 μg. per mg of microspheres.

EXAMPLE 11

Preparation of microspheres wherein the starting polymer is HYAFF-11 p75(benzyl ester of hyaluronic acid), and which contain GMCSF (granulocytemacrophage colony stimulating factor)

A hyaluronic acid ester, wherein 75% of the carboxy groups of hyaluronicacid are esterified with benzyl alcohol while the remaining part issalified with sodium, is dissolved in an aprotic solvent such asdimethylsulfoxide (DMSO), at a concentration varying between 0.1 and 5%in weight, generally 1% w/w. Once the polymer has reachedsolubilization. GMCSF is added to the polymeric solution at a setconcentration, eg 2% of the polymer mass.

The solution thus obtained is poured into a pressure-proof vessel(precipitator), thermostatically controlled by a heated ethylene glycoljacket. Porous steel filters with a cut-off of 0.1μ are screwed onto thetop and base of the precipitator. The liquid is unable to seep throughby gravity alone.

Once the vessel is closed, it is loaded from underneath with hyperpurecarbon dioxide (CO₂) until the working pressure is reached (80-120 bar,preferably 100 bar). The CO₂ is distributed in the solution through theporous base. This antisolvent, which is first gaseous and thensupercritical, can be mixed perfectly with the liquid solvent (DMSO) butit is a nonsolvent for the polymer and the polypeptide GMCSF.

The loading rate, or the pressure gradient over time, is set in a rangeof 3-20 bar/min, preferably 10 bar/min. The temperature in theprecipitator is kept constant in a range of between 25° C. and 60° C.,preferably 40° C.

When the working pressure has been reached, the flow of CO₂ is switchedoff for 10 minutes to obtain the desired pressure and temperatureconditions inside the precipitator. The washing operation is begun bysupplying antisolvent to the precipitator and regulating the outlet flowfrom the top of the precipitator by means of a millimetric valve.

The outlet fluid, constituted by antisolvent and DMSO, is directedtowards the DMSO collector, which is kept at room pressure; the DMSOseparates after expansion and consequent cooling, while the gaseous CO₂comes out of the top of the vessel and is released into the atmosphere.The solid particles, on the other hand, are trapped by the porousfilters at the top and base of the precipitator.

The operation is continued to allow the DMSO to be completely removedfrom the precipitator. The time it takes for the organic solvent to beremoved by the supercritical antisolvent depends on the temperature inthe precipitation chamber, when fixed amount of liquid solution andantisolvent flow rate are set up.

At the end of washing, the supply of CO₂ is cut off and the vessel isdepressurized at a rate of 5 bar/min. The vessel is opened, themicrospheres are collected and placed in suitable containers where theyare stored at 4° C. The yield of microspheres is almost total. There isno appreciable incorporation of solvent in the precipitate. The DMSO iscollected in the expansion container.

The mean particle size in these working conditions is 0.8μ. The quantityof incorporated GMCSF is 17 μg per mg of microspheres.

EXAMPLE 12

Preparation of microspheres wherein the starting polymer is HYAFF-7(ethyl ester of hyaluronic acid), and which are loaded with GMCSF(granulocyte macrophage colony stimulating factor)

A hyaluronic acid ester, wherein all the carboxy groups of hyaluronicacid are esterified with ethyl alcohol, is dissolved in an aproticsolvent such as dimethylsulfoxide (DMSO), at a concentration varyingbetween 0.1 and 5% in weight, generally 1% w/w. Once the polymer hasreached solubilization. GMCSF is added to the polymeric solution at aset concentration, eg 0.1% of the polymer mass.

The solution thus obtained is poured into a pressure-proof vessel(precipitator), thermostatically controlled by a heated ethylene glycoljacket. Porous steel filters with a cut-off of 0.1μ are screwed onto thetop and base of the precipitator. The liquid is unable to seep throughby gravity alone.

Once the vessel is closed, it is loaded from underneath with hyperpurecarbon dioxide (CO₂) until the working pressure is reached (80-120 bar,generally 100 bar). The CO₂ is distributed in the solution through theporous base. This antisolvent, which is first gaseous and thensupercritical, can be mixed perfectly with the liquid solvent (DMSO) butit is a nonsolvent for the polymer and the polypeptide GMCSF.

The loading rate, or the pressure gradient over time, is set in a rangeof 3-20 bar/min, preferably 10 bar/min. The temperature in theprecipitator is kept constant in a range of between 25° C. and 60° C.,preferably 40° C.

When the working pressure has been reached, the flow of CO₂ is switchedoff for 10 minutes to obtain the desired pressure and temperatureconditions inside the precipitator. The washing operation is begun bysupplying antisolvent to the precipitator and regulating the outlet flowfrom the top of the precipitator by means of a millimetric valve.

The outlet fluid, constituted by antisolvent and DMSO, is directedtowards the DMSO collector, which is kept at room pressure; the DMSOseparates after expansion and consequent cooling, while the gaseous CO₂comes out of the top of the vessel and is released into the atmosphere.The solid particles, on the other hand, are trapped by the porousfilters at the top and bottom of the precipitator.

The operation is continued to allow the DMSO to be completely removedfrom the precipitator. The time it takes for the organic solvent to beremoved by the supercritical antisolvent depends on the temperature inthe precipitation chamber, when fixed amount of liquid solution andantisolvent flow rate are set up.

At the end of washing, the supply of CO₂ is cut off and the vessel isdepressurized at a rate of 5 bar/min. The vessel is opened, themicrospheres are collected and placed in suitable containers where theyare stored at 4° C. The yield of microspheres is almost total. There isno appreciable incorporation of solvent in the precipitate. The DMSO iscollected in the expansion container.

The mean particle size in these working conditions is 1.0μ. The quantityof incorporated GMCSF is 0.9 μg per mg of microspheres.

EXAMPLE 13

Preparation of microspheres wherein the starting polymer is HYAFF-11(benzyl ester of hyaluronic acid), and which are loaded with humaninsulin

A hyaluronic acid ester, wherein all the carboxy groups of hyaluronicacid are esterified with benzyl alcohol, is dissolved in an aproticsolvent such as dimethylsulfoxide (DMSO), at a concentration varyingbetween 0.1 and 5% in weight, generally 1% w/w. Once the polymer hasreached solubilization, human insulin is added to the polymeric solutionat a set concentration, eg 5 I.U. per mg of polymer.

The solution thus obtained is poured into a pressure-proof vessel(precipitator), thermostatically controlled by a heated ethylene glycoljacket. Porous steel filters with a cut-off of 0.1μ are screwed onto thetop and base of the precipitator. The liquid is unable to seep throughby gravity alone.

Once the vessel is closed, it is loaded from underneath with hyperpurecarbon dioxide (CO₂) until the working pressure is reached (80-120 bar,preferably 100 bar). The CO₂ is distributed in the solution through theporous base. This antisolvent, which is first gaseous and thensupercritical, can be mixed perfectly with the liquid solvent (DMSO) butit is a nonsolvent for the polymer and the polypeptide human insulin.

The loading rate, or the pressure gradient over time, is set in a rangeof 3-20 bar/min, preferably 10 bar/min. The temperature in theprecipitator is kept constant in a range of between 25° C. and 60° C.,preferably 40° C.

When the working pressure has been reached, the flow of CO₂ is switchedoff for 10 minutes to obtain the desired pressure and temperatureconditions inside the precipitator. The washing operation is begun bysupplying antisolvent to the precipitator and regulating the outlet flowfrom the top of the precipitator by means of a millimetric valve.

The outlet fluid, constituted by antisolvent and DMSO, is directedtowards the DMSO collector, which is kept at room pressure; the DMSOseparates after expansion and consequent cooling, while the gaseous CO₂comes out of the top of the vessel and is released into the atmosphere.The solid particles, on the other hand, are trapped by the porousfilters at the top and bottom of the precipitator.

The operation is continued to allow the DMSO to be completely removedfrom the precipitator. The time it takes for the organic solvent to beremoved by the supercritical antisolvent depends on the temperature inthe precipitation chamber, when fixed amount of liquid solution andantisolvent flow rate are set up.

At the end of washing, the supply of CO₂ is cut off and the vessel isdepressurized at a rate of 5 bar/min. The vessel is opened, themicrospheres are collected and placed in suitable containers where theyare stored at 4° C. The yield of microspheres is almost total. There isno appreciable incorporation of solvent in the precipitate. The DMSO iscollected in the expansion container.

The mean particle size in these working conditions is 0.8μ. The quantityof incorporated insulin is 5 I.U. per mg of microspheres.

EXAMPLE 14

Surface loading of microspheres of HYAFF-11 (benzyl ester of hyaluronicacid) with calcitonin by lyophilization

Microspheres prepared according to Example 1 are suspended in a solutionof 0.01 M phosphate buffer, containing calcitonin in a concentrationwhich gives a protein titer of 1 I.U. per mg of suspended microspheres.After 15 minutes' shaking with a semiautomatic device, the container isimmersed in liquid nitrogen until the suspension is completely frozen.

Once frozen, the container is lyophilized for 24 hours, after which thelyophilized product is stored at 4° C.

The mean particle size in these working conditions is 0.4μ. The quantityof incorporated calcitonin is 1 I.U. per mg of microspheres.

EXAMPLE 15

Surface loading of microspheres of HYAFF-11 p75 (benzyl ester ofhyaluronic acid) with calcitonin by lyophilization

Microspheres prepared according to Example 2 are suspended in a solutionof 0.01 M phosphate buffer, containing calcitonin in a concentrationwhich gives a protein titer of 1.5 I.U. per mg of suspendedmicrospheres. After 15 minutes' stirring with a semiautomatic device,the container is immersed in liquid nitrogen until the suspension iscompletely frozen.

Once frozen, the container is lyophilized for 24 hours, after which thelyophilized product is stored at 4° C.

The mean particle size in these working conditions is 0.6μ. The quantityof incorporated calcitonin is 1.5 I.U. per mg of microspheres.

What is claimed is:
 1. Microspheres having a diameter ≧0.1 and <1μcomprising a a biocompatible acidic polysaccharide ester selected fromthe group consisting of hyaluronic acid esters, crosslinked esters ofhyaluronic acid.
 2. The microspheres according to claim 1 wherein saidester is the ethyl or benzyl ester of said polysaccharide.
 3. Themicrospheres according to claims 1, comprising the partial or totalbenzyl ester of hyaluronic acid.
 4. The microspheres according to claims1 further comprising at least once active principle selected from thegroup consisting of: a pharmaceutically active polypeptide, aGranulocyte Macrophage Colony Stimulating Factor (GMCSF), a trophicfactor, an immunoglobulin, a natural or a synthetic derivative of aganglioside, an antiviral, an antiasthmatic, an antiinflammatory agent,an antibiotic and an antimycotic agent.
 5. The microspheres according toclaim 4, wherein said pharmaceutically active polypeptide is selectedfrom the group consisting of calcitonin, insulin.
 6. The microspheresaccording to claim 4 wherein said trophic factor, is selected from thegroup consisting of: Nerve Growth Factor (h-NGF), Ciliary NeuronotrophicGrowth Factor (h-CNTF).
 7. The microspheres according to claim 1,consisting of said biocompatible acidic polysaccharide ester. 8.Pharmaceutical compositions administrable by oral, nasal, pulmonary,vaginal or rectal route, containing the microspheres according to claim1 as vehicling agents or carriers, in combination with at least oneactive principle and optionally other conventional excipients. 9.Pharmaceutical compositions administrable by oral, nasal, pulmonary,vaginal or rectal route, containing the microspheres according to claim4 optionally in combination with other excipients.
 10. Thepharmaceutical compositions according to claim 8 in a controlled releaseform, further containing suitable excipients normally used for preparingcontrolled release pharmaceutical compositions.
 11. Diagnosticscontaining the microspheres according to claim 1 as a tracer carrier.